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Development of a real-time PCR-based method for the measurement of neutralizing antibody to interferon-beta in multiple sclerosis patients

Background Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder of the central nervous system (CNS). In Hong Kong, the prevalence rates of MS is 4.8/100,000. First line disease modifying agent (DMA) type 1 interferon β (IFN-β) is the most commonly use therapy for relapsing and remitting MS(RRMS). Depending on the administration type and route of IFN-β, up to 80% of patients develop harmless binding antibody (BAb),which binds to IFN molecules but not necessary interfere its bioactivity. When IFN-β therapy continues, maturation of BAb response can lead to the formation of high affinity neutralizing antibody (NAb). About 45% of MS patients develop NAb against IFN-β in one year of IFN-β treatment. NAb shows a loss of IFN-β clinical effect by increasing MRI activity and disease progression. As the clinical effect of NAb is lagging behind the initial appearance of NAb in the body, it is suggested to develop a NAb assay to predict treatment failure and advice switching therapy for patients when NAb is present.

Aim The aim of this study was:
I. To develop and evaluate a qPCR-based method for the measurement of NAb to IFN-β in MS patient.
II. To establish the normal reference range of NAb in Chinese population.
III. To seek the possibility of using anti-IFN-β BAb assay and in vivo MxA gene expression assay as a screening test for NAb
IV. To compare the performance between MxA induction qPCR, ELISA, WB assay and luciferase IFN-β reporter gene assay

Materials and methods23Chinese RRMS patients who treated with IFN-β-1a therapy for a minimum of12 months were recruited in this study. Serum and PBMC were collected12 hours after the IFN-β-1a injection. MxA, IFNAR1 and IFNAR2 mRNA from PBMC were tested byin vivo MxA gene expression assay. NAb containing serum was tested by anti-IFN-β BAb assay, IFN-β reporter gene assay, in vitro MxA induction WB, ELISA and qPCR assay. In addition, blood samples from 3 Chinese volunteers without any known autoimmune disease history were collected to evaluate the baseline of NAb titer and MxA expression.

Result The experimental condition of MxA induction qPCR assay was optimized by using 2.5×105A549 cells plating density, 10% FCS concentration,5 hours IFN-β stimulation time and GAPDH normalization. Assay accuracy was validated by reference anti-IFN-β antibody. Starting from 2.5 TRU, linear relationship could be observed (r2= 0.9873). The lower limit of quantification (LLOQ) was 0.02 LU/mL, the upper limit of quantification (ULOQ) was 16LU/mL and the limit of detection (LOD) was 0.002 LU/mL. The reproducibility of the assay was measured, the intra-and inter-assay imprecision(%CV)for high value were 5.95% and 7.17% respectively, while the intra-and inter-assay impression were8.31% and 15.95%respectively.Results of the qPCR-based method were concurring with that of luciferase IFN-β reporter gene assay. The upper limit of the NAb reference range in Chinese population was 40.3 TRU (n=3, 95% CI = 31.7-48.8). The performance observed in MxA induction ELISA assay swas unsatisfactory. The correlation of anti-IFN-β BAb assay and in vivoMxA gene expression assay results with NAb status indicated both tests were sensitive enough for NAb screening.

Conclusion A normal range of NAb titer in Chinese population was established in this study. Anti-IFN-β BAb assay and in vivo MxA gene expression assay were proved suitable for NAb screening. The performance of the developed MxA induction qPCR assay was superior to MxA induction ELISA, WB assay and comparable to luciferase IFN-β reporter gene assay. By using MxA induction qPCR assay, actual efficacy of IFN-β therapy could be measured and monitored. Any treatment failure could be predicted earlier. / published_or_final_version / Pathology / Master / Master of Medical Sciences
Date January 2014
CreatorsLeung, Chieh-wing, Jervis, 梁倢榮
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Source SetsHong Kong University Theses
Detected LanguageEnglish
RightsCreative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works.
RelationHKU Theses Online (HKUTO)

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